Proteomic analysis of kidneys from selenoprotein M transgenic rats in response to increased bioability of selenium

Background

To characterize changes in global protein expression in kidneys of transgenic rats overexpressing human selenoprotein M (SelM) in response to increased bioabivility of selenium (Sel), total proteins extracted from kidneys of 10-week-old CMV/hSelM Tg and wild-type rats were separated by 2-dimensional gel electrophoresis and measured for changes in expression.

Results

Ten and three proteins showing high antioxidant enzymatic activity were up- and down-regulated, respectively, in SelM-overexpressing CMV/hSelM Tg rats compared to controls based on an arbitrary 2-fold difference. Up-regulated proteins included LAP3, BAIAP2L1, CRP2, CD73 antigen, PDGF D, KIAA143 homolog, PRPPS-AP2, ZFP313, HSP-60, and N-WASP, whereas down-regulated proteins included ALKDH3, rMCP-3, and STC-1. After Sel treatment, five of the up-regulated proteins were significantly increased in expression in wild-type rats, whereas there were no changes in CMV/hSelM Tg rats. Only two of the down-regulated proteins showed reduced expression in wild-type and Tg rats after Sel treatment.

Conclusions

These results show the primary novel biological evidences that new functional protein groups and individual proteins in kidneys of Tg rats relate to Sel biology including the response to Sel treatment and SelM expression.     

Cancer Risks in Parents Who had a Child with a Congenital Malformation

Cancer risk in parents may be related to congenital malformations (CMs) in their children if they share genetic susceptibility or environmental exposure that may be teratogenic and carcinogenic. We conducted a population‐based cohort study based on Danish register data. We identified 795,607 mothers and 781,424 fathers who had all their children between 1977 and 2007 in Denmark. Information on CM was obtained from the Danish Hospital Registry and information on cancer was obtained from the Danish Cancer Registry. Parents were followed from the birth of their first child until the diagnosis of cancer, death, emigration, or December 31, 2007. We used Cox regression models to estimate hazard ratios (HRs) for cancer including cancer in specific organs in mothers and fathers. Overall, 75,701 (9.5%) mothers and 72,724 (9.3%) fathers had at least one child diagnosed with CMs within the first year of life. Neither mothers (HR = 1.04; 95% CI: 0.99–1.04) nor fathers (HR = 1.03; 95% CI: 0.98–1.09) who had a child with a CM had a higher overall risk of cancer. Mothers (HR = 0.76, 95% CI: 0.58–1.00) or fathers (HR = 0.89, 95% CI: 0.66–1.19) who had a child with a chromosomal malformation had a lower overall cancer risk. The findings also showed a higher risk for some specific types of cancer in parents who had children with a CM in the specific system. Some, or perhaps all, of these findings may be due to chance caused by multiple comparisons. We present all results on paper or online to provide clues for further research and to avoid publication bias.     

Generation of Five Human Lactoferrin Transgenic Cloned Goats Using Fibroblast Cells and Their Methylation Status of Putative Differential Methylation Regions of IGF2R and H19 Imprinted Genes

Background

Somatic cell nuclear transfer (SCNT) is a promising technique to produce transgenic cloned mammalian, including transgenic goats which may produce Human Lactoferrin (hLF). However, success percentage of SCNT is low, because of gestational and neonatal failure of transgenic embryos. According to the studies on cattle and mice, DNA methylation of some imprinted genes, which plays a vital role in the reprogramming of embryo in NT maybe an underlying mechanism.

Methodology/Principal Findings

Fibroblast cells were derived from the ear of a two-month-old goat. The vector expressing hLF was constructed and transfected into fibroblasts. G418 selection, EGFP expression, PCR, and cell cycle distribution were applied sequentially to select transgenic cells clones. After NT and embryo transfer, five transgenic cloned goats were obtained from 240 cloned transgenic embryos. These transgenic goats were identified by 8 microsatellites genotyping and southern blot. Of the five transgenic goats, 3 were lived after birth, while 2 were dead during gestation. We compared differential methylation regions (DMR) pattern of two paternally imprinted genes (H19 and IGF2R) of the ear tissues from the lived transgenic goats, dead transgenic goats, and control goats from natural reproduction. Hyper-methylation pattern appeared in cloned aborted goats, while methylation status was relatively normal in cloned lived goats compared with normal goats.

Conclusions/Significance

In this study, we generated five hLF transgenic cloned goats by SCNT. This is the first time the DNA methylation of lived and dead transgenic cloned goats was compared. The results demonstrated that the methylation status of DMRs of H19 and IGF2R were different in lived and dead transgenic goats and therefore this may be potentially used to assess the reprogramming status of transgenic cloned goats. Understanding the pattern of gene imprinting may be useful to improve cloning techniques in future.     

Rapid Identification of Antifungal Compounds against Exserohilum rostratum Using High Throughput Drug Repurposing Screens

A recent large outbreak of fungal infections by Exserohilum rostratum from contaminated compounding solutions has highlighted the need to rapidly screen available pharmaceuticals that could be useful in therapy. The present study utilized two newly-developed high throughput assays to screen approved drugs and pharmaceutically active compounds for identification of potential antifungal agents. Several known drugs were found that have potent effects against E. rostratum including the triazole antifungal posaconazole. Posaconazole is likely to be effective against infections involving septic joints and may provide an alternative for refractory central nervous system infections. The anti-E. rostratum activities of several other drugs including bithionol (an anti-parasitic drug), tacrolimus (an immunosuppressive agent) and floxuridine (an antimetabolite) were also identified from the drug repurposing screens. In addition, activities of other potential antifungal agents against E. rostratum were excluded, which may avoid unnecessary therapeutic trials and reveals the limited therapeutic alternatives for this outbreak. In summary, this study has demonstrated that drug repurposing screens can be quickly conducted within a useful time-frame. This would allow clinical implementation of identified alternative therapeutics and should be considered as part of the initial public health response to new outbreaks or rapidly-emerging microbial pathogens.     

Different Distribution of HIV-1 Subtype and Drug Resistance Were Found among Treatment Na?ve Individuals in Henan,Guangxi, and Yunnan Province of China

Yunnan, Guangxi and Henan are the provinces with the most severe HIV epidemic in China, which were also among the first group of areas providing free ART in 2004. However, little comprehensive data are available on prevalence of HIV subtype and baseline drug resistance in drug-naïve populations. In this study, 1746 treatment-naïve HIV-positive individuals were randomly selected from new-reported cases in Henan, Guangxi and Yunnan. Among of them, subtypes and drug resistance of 1159 strains were determined by amplifying and sequencing full-length pol genes. Significantly different distributions of HIV subtypes prevalent in three provinces were identified (P<0.01). CRF08_BC was found dominant in Yunnan (59.8%), while CRF01_AE was dominant in Guangxi (77.3%) and subtype B was dominant in Henan province (93.9%). The total prevalence of drug resistance was 7.1%. The highest prevalence of HIV drug resistance was found in Henan (12.2%), followed by Yunnan (5.6%) and Guangxi (3.3%). The results of this study suggest that genetic drug-resistance should be tested before initiation of ART in China, especially in Henan province. Furthermore, the prevalence of HIV drug resistant strains should be considered separately in different areas in China before the change of different free ART regimens.     

Gene Expression-Based Classifiers Identify Staphylococcus aureus Infection in Mice and Humans

Staphylococcus aureus causes a spectrum of human infection. Diagnostic delays and uncertainty lead to treatment delays and inappropriate antibiotic use. A growing literature suggests the host’s inflammatory response to the pathogen represents a potential tool to improve upon current diagnostics. The hypothesis of this study is that the host responds differently to S. aureus than to E. coli infection in a quantifiable way, providing a new diagnostic avenue. This study uses Bayesian sparse factor modeling and penalized binary regression to define peripheral blood gene-expression classifiers of murine and human S. aureus infection. The murine-derived classifier distinguished S. aureus infection from healthy controls and Escherichia coli-infected mice across a range of conditions (mouse and bacterial strain, time post infection) and was validated in outbred mice (AUC>0.97). A S. aureus classifier derived from a cohort of 94 human subjects distinguished S. aureus blood stream infection (BSI) from healthy subjects (AUC 0.99) and E. coli BSI (AUC 0.84). Murine and human responses to S. aureus infection share common biological pathways, allowing the murine model to classify S. aureus BSI in humans (AUC 0.84). Both murine and human S. aureus classifiers were validated in an independent human cohort (AUC 0.95 and 0.92, respectively). The approach described here lends insight into the conserved and disparate pathways utilized by mice and humans in response to these infections. Furthermore, this study advances our understanding of S. aureus infection; the host response to it; and identifies new diagnostic and therapeutic avenues.     

Hybrid Adeno-Associated Viral Vectors Utilizing Transposase-Mediated Somatic Integration for Stable Transgene Expression in Human Cells

Recombinant adeno-associated viral (AAV) vectors have been shown to be one of the most promising vectors for therapeutic gene delivery because they can induce efficient and long-term transduction in non-dividing cells with negligible side-effects. However, as AAV vectors mostly remain episomal, vector genomes and transgene expression are lost in dividing cells. Therefore, to stably transduce cells, we developed a novel AAV/transposase hybrid-vector. To facilitate SB-mediated transposition from the rAAV genome, we established a system in which one AAV vector contains the transposon with the gene of interest and the second vector delivers the hyperactive Sleeping Beauty (SB) transposase SB100X. Human cells were infected with the AAV-transposon vector and the transposase was provided in trans either by transient and stable plasmid transfection or by AAV vector transduction. We found that groups which received the hyperactive transposase SB100X showed significantly increased colony forming numbers indicating enhanced integration efficiencies. Furthermore, we found that transgene copy numbers in transduced cells were dose-dependent and that predominantly SB transposase-mediated transposition contributed to stabilization of the transgene. Based on a plasmid rescue strategy and a linear-amplification mediated PCR (LAM-PCR) protocol we analysed the SB100X-mediated integration profile after transposition from the AAV vector. A total of 1840 integration events were identified which revealed a close to random integration profile. In summary, we show for the first time that AAV vectors can serve as template for SB transposase mediated somatic integration. We developed the first prototype of this hybrid-vector system which with further improvements may be explored for treatment of diseases which originate from rapidly dividing cells.     

Extracellular HSP70/HSP70-PCs Promote Epithelial-Mesenchymal Transition of Hepatocarcinoma Cells

Background

Extracellular heat shock protein 70 and peptide complexes (eHSP70/HSP70-PCs) regulate a variety of biological behaviors in tumor cells. Whether eHSP70/HSP70-PCs are involved in the epithelial-mesenchymal transition (EMT) of tumor cells remains unclear.

Aims

To determine the effects of eHSP70/HSP70-PCs on EMT of hepatocarcinoma cells.

Methods

The expressions of E-cadherin, HSP70, α-smooth muscle actin protein (α-SMA) and p-p38 were detected immunohistochemically in liver cancer samples. Immunofluorescence, western blotting and real-time RT-PCR methods were used to analyze the effects of eHSP70/HSP70-PCs on the expressions of E-cadherin, α-SMA and p38/MAPK in vivo.

Results

HSP70, E-cadherin, α-SMA and p-p38 were elevated in hepatocellular carcinoma tissues. The expression of HSP70 was positively correlated with malignant differentiated liver carcinoma. The expressions of HSP70, α-SMA and p-p38 correlated with recurrence-free survival after resection. eHSP70/HSP70-PCs significantly promoted the expressions of α-SMA and p-p38 and reduced the expressions of E-cadherin in vivo. The effect was inhibited by SB203580.

Conclusion

The expressions of HSP70, E-cadherin, α-SMA and p-p38 may represent indicators of malignant potential and could discriminate the malignant degree of liver cancer. eHSP70/HSP70-PCs play an important role in the EMT of hepatocellular carcinoma via the p38/MAPK pathway.